14

Glycolysis

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14-1
14 The Glycolytic Pathway
Figure 14.1 Glucose is converted to two pyruvate

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14-2
14 Glycolysis
• Glycolysis: a series of 10 enzyme-catalyzed
reactions by which glucose is oxidized to two
molecules of pyruvate
O
glycolysis
C6 H1 2 O6 2 CH3 CCOO - + 2 H+
Glucose Pyruvate

• there is net conversion of 2ADP to 2ATP

O
C6 H1 2 O6 + 2 ADP + 2 Pi 2 CH3 CCOO - + 2 ATP
Glucose Pyruvate

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14-3
14 Fates of Pyruvate
• Pyruvate is most commonly metabolized in one of
three ways, depending on the type of organism
and the presence or absence of O2
aerobic conditions
plants and animals 3 CO 2 + 2 H2 O

O OH
anaerobic conditions
CH3 CCOO - CH3 CHCOO-
contracting muscle
Pyruvate Lactate
anaerobic conditions
CH3 CH2 OH + CO 2
fermentation in yeast
Ethanol

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14-4
14 Glycolysis - Rexn 1
• Reaction 1: phosphorylation of -D-glucose
CH2 OH
HO O O O
HO hexokinase
+ - O- P-O- P-O- AMP 2+
OH Mg
O- O-
-D-Glucose OH ATP

CH2 OPO 3 2 -
HO O O
HO
+ - O- P-O- AMP
OH O-
OH
-D-Glucose-6-phosphate ADP

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14-5
14 Glycolysis - Rexn 1
• this reaction is driven by the free energy of hydrolysis
of ATP
Glucose + P i Glucose-6-phosphate + H2 O G o' = +13.8 kJ•mol -1

ATP + H2 O AD P + Pi G o' = -30.5 kJ•mol -1

Glucose + ATP Glucose-6-phosphate + AD P G o' = -16.7 kJ•mol -1

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14 Glycolysis - Rexn 1
• catalyzed by hexokinase
• a large conformational change takes place when
substrate is bound to the enzyme
Figure 14.4 Hexokinase and hexokinase-glucose complex

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14 Glycolysis - Rexn 2
• Reaction 2: isomerization of glucose-6-phosphate
to fructose-6-phosphate
6 6
CH2 OPO 3 2- CH2 OPO 3 2 - 1
phosphogluco- CH2 OH
HO O O
isomerase
HO H HO 2
2 1
OH H OH
OH HO H
-D-Glucose-6-phosphate -D-Fructose-6-phosphate

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14-8
14 Glycolysis - Rexn 2
• this isomerization is most easily seen by considering
the open-chain forms of each monosaccharide; it is one
keto-enol tautomerism followed by another
1 1 CH
CHO H C OH 2 OH
2 2C
H OH C OH O
HO H HO H HO H
H OH H OH H OH
H OH H OH H OH
CH2 OPO 3 2 - CH2 OPO 3 2 - CH2 OPO 3 2 -
Glucose-6-phosphate (An enediol) Fructose-6-phosphate

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14-9
14 Glycolysis - Rexn 3
• Reaction 3: phosphorylation of fructose-6-
phosphate
6
CH2 OPO 3 2 - 1
O CH2 OH phospho-
H HO fructokinase
+ A TP
H OH Mg 2 +
HO H
6
-D-Fructose-6-phosphate CH2 OPO 3 2 - 1
O CH2 OPO 3 2 -
H HO + A DP
H OH
HO H
-D-Fructose-1,6-bisphosphate

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14-10
14 Glycolysis - Rexn 3
• Phosphofructokinase: a key regulatory enzyme in
the metabolism of glucose
• a tetramer and subject to allosteric feedback
• the tetramer is composed of L and M subunits
• M4, M3L, M2L2, ML3, and L4 all exist
• muscles are rich in M4; the liver is rich in L4
• ATP is an allosteric effector; high levels inhibit the
enzyme, low levels activate it
• fructose-1,6-bisphosphate is also an allosteric effector

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14 Glycolysis - Rexn 3
Figure 14.6 Phosphofructokinase is an allosteric enzyme

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14 Glycolysis - Rexn 4
• Reaction 4: cleavage of fructose-1,6-bisphosphate
to two triose phosphates
2- CH2 OPO 3 2 -
CH2 OPO 3
C= O Dihydroxyacetone
C= O phosphate
aldolase CH2 OH
HO H
H OH CHO
H OH D-Glyceraldehyde
H C OH
3-phosphate
CH2 OPO 3 2 -
CH2 OPO 3 2 -
Fructose-1,6-bisphosphate

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14-13
14 Glycolysis - Rexn 5
• Reaction 5: isomerization of triose phosphates
• catalyzed by triosephosphate isomerase
• reaction involves two successive keto-enol
tautomerizations
• only the D enantiomer of glyceraldehyde 3-phosphate is
formed
CH2 OH CHOH CHO
C= O C-OH H C OH
CH2 OPO 3 2 - CH2 OPO 3 2 - CH2 OPO 3 2 -
Dihydroxyacetone An enediol D-Glyceraldehyde
phosphate intermediate 3-phosphate

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14-14
14 Glycolysis - Rexn 6
• Reaction 6: oxidation of the -CHO group of D-
glyceraldehyde-3-phosphate
• the -CHO group is oxidized to a carboxyl group
• the oxidizing agent, NAD+, is reduced to NADH
A two-electron oxidation
O O
G- C- H + H2 O G- C- OH + 2 H+ + 2 e -

A two-electron reduction
N AD + + H+ + 2 e - N AD H

O O
G- C- H + H2 O + N AD + G- C- OH + H+

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14-15
14 Glycolysis - Rexn 6
• We divide this reaction into three steps
• step 1: formation of a thiohemiacetal
O OH
G- C- H + HS-Enz G- C- S- Enz
H
Glyceraldehyde A thiohemiacetal
3-phosphate

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14-16
14 Glycolysis - Rexn 6
• step 2: oxidation of the thiohemiacetal by NAD+
an enzyme-bound
H thioester
O O
G- C- S- Enz G- C- S- Enz
H O H H O
CNH 2
CNH 2

N+

:
N
Ad Ad

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14-17
14 Glycolysis - Rexn 6
• step 3: conversion of the thioester to a mixed anhydride
O O
-
G- C- S- Enz + O- P-OH
O-

O- O O O
G- C- O-P- OH G- C- O-P- O - + Enz -S-
Enz- S O- O-
A tetrahedral 1,3-Bisphosphoglycerate
carbonyl addition (a mixed anhydride)
intermediate

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14-18
14 Glycolysis - Rexn 6
• the overall reaction involves an exergonic oxidation
and an endergonic phosphorylation
• the overall reaction is slightly endergonic
O O
oxidation: C-H to C-O - G o' = -43.1 kJ•mol -1

O O O
phosphorylation: C-O - to C-O- P-O - G o' = +49.3 kJ•mol -1

O-

O O O
C-H to C-O- P-O - G o' = +6.2 kJ•mol -1

O-

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14-19
14 Glycolysis - Rexn 7
• Reaction 7: transfer of a phosphate group from
1,3-bisphosphoglycerate to ADP
• this reaction is called substrate-level phosphorylation
O phospho-
C-OPO 3 2 - O glycerate kinase
H C OH + - O- P-O- AMP
CH2 OPO 3 2 - O- Mg 2+
1,3-Bisphospho-
glycerate ADP
COO- O O
H C OH + - O- P-O- P-O- AMP
CH2 OPO 3 2 - O- O-
3-Phosphoglycerate A TP

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14-20
14 Glycolysis - Rexn 7
• this reaction is the sum of the endergonic
phosphorylation of ADP and the exergonic hydrolysis
of the mixed phosphate anhydride
phosphorylation:
AD P + Pi ATP + H2 O G o' = +0.5 kJ•mol -1

hydrolysis:
O O O
C-O- P-O - + H2 O C-O - + Pi G o' = -49.3 kJ•mol -1

O-
O O O
C-O- P-O - + AD P + Pi C-O - + ATP G o' = -18.8 kJ•mol -1

O-

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14-21
14 Glycolysis - Rexn 8
• Reaction 8: isomerization of 3-phosphoglycerate
to 2-phosphoglycerate
COO- phosphoglycerate COO-
mutase
H C OH H C OPO 3 2 -
CH2 OPO 3 2 - CH2 OH
3-Phosphoglycerate 2-Phosphoglycerate

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14-22
14 Glycolysis - Rexn 9
• Reaction 9: dehydration of 2-phosphoglycerate
COO- COO-
2-
enolase
H C OPO 3 C OPO 3 2 - + H2 O
Mg 2+
CH2 OH CH2
2-Phosphoglycerate Phosphoenolpyruvate

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14-23
14 Glycolysis - Rexn 10
• Reaction 10: phosphate transfer to ADP
• stage 1: transfer of the phosphate group
COO- O
pyruvate
kinase
C OPO 3 2 - + - O- P-O- AMP
Mg 2+
CH2 O-
Phosphoenol- ADP
pyruvate COO- O O
C-OH + - O- P-O- P-O- AMP
CH2 O- O-
ATP
Enol of
pyruvate

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14-24
14 Glycolysis - Rexn 10
• stage 2: enolization to pyruvate
COO- COO-
C-OH C= O
CH2 CH3
Enol of pyruvate Pyruvate

• reaction 10 is the sum of an exergonic hydrolysis and

an endergonic phosphorylation of ATP
hydrolysis
PEP + H2 O Pyruvate + Pi G o' = -61.9 kJ•mol -1

phosphorylation
AD P + Pi ATP + H2 O G o' = +30.5 kJ•mol -1

PEP + AD P Pyruvate + ATP G o' = -31.4 kJ•mol -1

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14-25
14 Glycolysis
• Summing these 10 reactions gives the net
equation for glycolysis

glycolysis
C6 H1 2 O6 + 2 N A D+ + 2 HPO 4 2 - + 2 A DP
Glucose
O
2 CH3 CCOO - + 2 NADH + 2 ATP + 2 H 2 O + 2 H +
Pyruvate

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14-26
14 Energetics of Glycolysis
• The free energy change, G°’, for the 10 reactions
of glycolysis are variously negative and positive
but, taken together, occur with a large decrease in
free energy
• Three reactions exhibit particularly large
decreases in free energy; the enzymes that
catalyze these reactions are sites of allosteric
control
• hexokinase
• phosphofructokinase
• pyruvate kinase

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14-27
14 Energetics of Glycolysis
• hexokinase G°'
(kJ•mol -1)
Glucose + Pi Glucose-6-P + H2 O +13.8
ATP + H2 O ADP + Pi -30.5

Glucose + ATP Glucose-6-P + ADP -16.7

• phosphofructokinase G°'
(kJ•mol -1)
Fructose-6-P + Pi Fructose-1,6-DiP + H2 O +16.7
ATP + H2 O ADP + Pi -30.5

Glucose + ATP+ ATP →

Glucose-6-phosphate + ADP -13.8
Fructose-6-P Fructose-1,6-DiP + ADP -13.8

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14-28
14 Energetics of Glycolysis
• pyruvate kinase
G°'
(kJ•mol -1)
Phosphoenolpyruvate + H2 O Pyruvate + Pi -61.9
ADP + Pi ATP + H2 O +30.5

Phosphoenolpyruvate + ADP Pyruvate + ATP -31.4

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14-29
14 Energetics of Glycolysis
• conversion of 1,3-bisphosphoglycerate (1,3-bisPG) to 3-
phosphoglycerate also occurs with a large decrease in
free energy
G°'
(kJ•mol -1)
1,3-bisPG + H2 O 3-Phosphoglycerate + Pi -49.3
ADP + Pi ATP + H2 O +30.5

1,3-bisPG + ADP 3-Phosphoglycerate + ATP -18.8

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14-30
14 Reactions of Pyruvate
• Pyruvate does not accumulate in cells, but rather
undergoes one of three enzyme-catalyzed
reactions, depending of the type of cell and its
state of oxygenation
• reduction to lactate
• reduction to ethanol
• oxidation and decarboxylation to acetyl-CoA
• A key to understanding the biochemical logic
behind two of these fates is to recognize that
glycolysis needs a continuing supply of NAD+
• if no oxygen is present to reoxidize NADH to NAD+, then
another way must be found to reoxidize it
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14-31
14 Lactate Fermentation
• In vertebrates under anaerobic conditions, the
most important pathway for the regeneration of
NAD+ is reduction of pyruvate to lactate
O lactate
dehydrogenase
CH3 CCOO - + NA DH + H +
Pyruvate
OH
CH3 CHCOO- + NA D+
Lactate

• lactate dehydrogenase (LDH) is a tetrameric isoenzyme

consisting of H and M subunits; H4 predominates in
heart muscle, and M4 in skeletal muscle
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14-32
14 Pyruvate to Lactate
• while lactate fermentation allows glycolysis to
continue, it increases the concentration of lactate and
also of H+ in muscle tissue
lactate OH
fermentation
C6 H1 2 O6 2 CH3 CHCOO- + 2 H+
Glucose Lactate
• when blood lactate reaches about 0.4 mg/100 mL,
muscle tissue becomes almost completely exhausted

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14-33
14 Glucose to Lactate
• Lactate fermentation occurs with a significant
decrease in free energy
G°'
(kJ•mol- 1)
+
Glucose + 2ADP + 2P i + 2NAD -73.3
2Pyruvate + 2ATP + 2NADH
+ +
2Pyruvate + 2NADH + 2H 2Lactate + 2NAD -50.2

Glucose + 2ADP + 2P i 2Lactate + 2ATP -123.5

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14-34
14 Pyruvate to Ethanol
• Yeasts and several other organisms regenerate
NAD+ by this two-step pathway
• decarboxylation of pyruvate to acetaldehyde
pyruvate O
O decarboxylase
- +
CH3 CCOO + H CH3 CH + CO 2
Pyruvate Acetaldehyde
• reduction of acetaldehyde to ethanol
alcohol
O
dehydrogenase
+
CH3 CH + N AD H + H
Acetaldehyde CH3 CH2 OH + NA D +
Ethanol

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14-35
14 Pyruvate to Acetyl-CoA
• Under aerobic conditions, pyruvate undergoes
oxidative decarboxylation
• the carboxylate group is converted to CO2
• the remaining two carbons are converted to the acetyl
group of acetyl-CoA
O oxidative
decarboxylation
CH3 CCOO - + NA D+ + CoA SH
Pyruvate
O
CH3 CSCoA + CO2 + N A DH
Acetyl-CoA

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14-36
14 Pyruvate to Acetyl-CoA
• oxidative decarboxylation of pyruvate to acetyl-CoA is
considerably more complex than the previous equation
suggests
• in addition to NAD+ and coenzyme A, it also requires
FAD, thiamine, and lipoic acid
H3 C CH2 CH2 OH
CH2 CH2 CH2 CH2 COO -
N H2
N S
N + S S H Lipoic acid
H (shown as the
H3 C N
Thiamine carboxylate anion)

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